TGF-ß signaling and cardiovascular progenitor cells for regenerative medicine

 

Cardiomyocyte progenitor cells to repair the injured heart

After a myocardial infarction, approximately one billion heart muscle cells die and need to be replace to preserve heart function. Until very recently, it was generally accepted that postnatal cardiac muscle was incapable of regeneration. The generally accepted paradigm was that adult cardiomyocytes lack the ability to regenerate the myocardium because they proliferate only up to the time of birth. Repair of the injured heart therefore results in replacement of resident cardiomyocytes by fibroblasts and the formation of fibrotic tissue to prevent rupture and dilatation.

Using stem cells to generate new cardiomyocytes for cell transplantation is an attractive approach. Stem cells are characterized by their self-renewal and multilineage differentiation capacity at a single cell level. Different types of stem cells exist. Embryonic stem cells are derived from a blastocyst stage embryo. These cells are pluripotent, and when introduced into a morula or blastocyst, can contribute to all three germlayers (ectoderm, endoderm, and mesoderm). Cardiomyocyte differentiation from ES cells and EC cells has been well described. Adult mesenchymal stem cells, isolated from bone marrow, peripheral or umbilical cord blood or adipose tissue, are thought to have a more limited differentiation capacity. But, they still can differentiate to a wide variety of cell types, in particular bone, cartilage, fat, and muscle cells. The biological function of adult stem cells is most likely to participate in regeneration and repair after for example myocardial infarction. Adult bone marrow and haematopoietic stem cells have been successfully engrafted into ischemic hearts, and reported to improve its function. But the predominant in vivo effect of BM or endothelial progenitor cells may be neoangiogenesis or arteriogenesis, not cardiomyocyte differentiation, underscoring the need to look for better stem cell sources for cardiomyocyte replacement.

The search for a cardiac stem or progenitor cell was considered futile given the accepted lack of regenerative potential of the heart. However, we were able to isolate cardiac progenitor cells using human heart tissue and developed a protocol that allows their efficient differentiation into cardiomyocytes in vitro without the need for co-culture with neonatal cardiomyocytes. These CMPCs provide us with the opportunity to unravel the signaling cascades involved in human cardiomcyotes differentiation as well as human cardiomyocyte biology.

Much remains to be understood before stem cell-based therapies can be effectively used for cardiac repair. Knowledge about the signaling pathways that steer the processes of self-renewal and controlled differentiation is critical to fully exploit the clinical use of cardiovascular progenitor cells. Little is known about the in vivo signals that orchestrate these events. Members of the transforming growth factor beta (TGF-ß) super family of growth factors, which includes TGF-ß , bone morphogenetic proteins (BMPs), and activins, are secreted proteins that have important roles in embryonic development, in particular in directing cell fate. ES cells are directed to differentiate into cardiomyocytes by signals mediated through TGF-ß and/or BMP, both in vitro as well as in vivo. The molecular signals that induce commitment, proliferation, and differentiation of stem cells into cardiomyocytes within an infracted myocardium are still not known, but a signaling pathway, like TGF-ß and/or BMP, might be required for therapeutic benefit of stem cell transplantation in the diseased heart.

There are several questions we need to address before we can be confident in using stem cells for cardiac repair.

  1. How important are stem cells with respect to the formation of new cardiomyocytes, or are stem cells only little growth factor bullets, producing growth factors, like TGF-ß, to support survival and repair of the damaged tissue?
  2. What are the signals in the host environment that stem cells respond to and induce their differentiation towards cardiomyocytes?
  3. What is the best route to transplant cells? Do we inject the stem cells into the hostile environment of the injured heart, or should we transplant heart tissue that was generated in a dish?

TGF-ß signaling in tissue repair.

One of the most extensively characterized biological functions of TGF-ß is its role in regulating physiological and pathological inflammation and fibrosis. TGF-ß affects virtually all stages of the chronic inflammation and fibrotic disease process. It will attract inflammatory cells and fibroblasts, it activates neutrophils and it induces cytokine production by macrophages, one of which is TGF-ß itself. TGF-ß is able to induce extracellular matrix deposition by stimulating the production of procollagen and fibronectin by fibroblasts, and down regulating the expression of proteases and upregulating protease inhibitors like plasminogen activator inhibitor type I (PAI-I) and tissue inhibitor of metalloproteinase-1 (Timp-1).

TGF-ß s transduce their signal via a specific complex of type I and type II serine/threonine kinase receptors. Upon ligand binding, the TGF-ß type II receptor recruits and phosphorylates the type I receptor. The type I receptor, also termed activin receptor like kinase (ALK), acts downstream of the type II receptor and propagates the signal to the nucleus by phosphorylating specific receptor regulated (R-)Smads. Smad2 and Smad3 are phosphorylated by the TGF-ß type I receptor, also known as activin receptor-like kinase 5 (ALK5). Once phosphorylated, the R-Smad heterodimerizes with the common mediator Smad4 and this complex translocates to the nucleus to regulate transcription. Smad2 and Smad3 are highly homologues, but have distinct modes of action. Whereas Smad2 can not bind DNA directly and regulates transcription by binding to other DNA-binding transcription factors, Smad3 regulates the activity of its targets genes by direct binding to DNA. Many genes, such as the ECM protein collagen type I, and their induction by TGF-ß , has been shown to be Smad3 dependent.

TGF-ß signalling plays an important role in cardiac development, cardiac hypertrophy, ventricular remodelling and the early response to myocardial infarction. During the early phase after myocardial infarction, there is an inflammatory response, followed by cell death and scar formation (collagen deposition) in the infarct zone and the development of fibrosis in non-infarcted segments of the myocardium in order to repair the damage caused by a loss of cardiomyocytes. At the site of myocardial injury, TGF-ß levels are elevated, resulting in an increase in inflammation, apoptosis and extracellular matrix deposition, ultimately leading to fibrosis. We want to inhibit the TGF-ß pathway by knocking out specific components of the cascade and analyse the effect this has on cardiomyocyte loss, inflammation and fibrosis and how if this result in improved recovery of the heart after myocardial infarction.